(A) FACS analyses showing human resting T lymphocytes from healthy donor or THP-1 monocytes were stained for surface (left panels) and total (after cell permeabilization, right panels) CCR5 (upper panels) or CD4 (lower panels), using 1/85a anti-human CCR5 antibody or OKT4 anti-human CD4, respectively, and analysed by flow cytometry. Green and red histograms correspond to specific labelling for CCR5 and CD4, respectively. Gray histograms are the isotypic control for each antibody. (B) Human resting T lymphocytes, THP-1 and Jurkat cells (negative control) were stained for surface (non-permeabilized, NP) and total (permeabilized P) CCR5 or CD4, using 2D7 anti-human CCR5 or OKT4 anti-human CD4 antibodies. Images were obtained by confocal microscopy. Scale bar: 10μM. (C) THP-1 cells, expressing endogenous receptor, were stained with the 2D7 anti-human CCR5 antibody and antibodies directed against Bip, an ER marker, or Giantin, a Golgi marker (see methods), and analysed by confocal microscopy. Colocalization of CCR5 (green) with Bip or Giantin (both in red) is shown in orange (Merge). Scale bar: 10μM.

CHO-K1 cells were transfected with the plasmids coding for CD4-Luc and HA-CCR5-YFP or HA-CCR5ΔCter-YFP at concentrations yielding sub-maximal BRET (indicated by an asterisk in figure 2E). Fourteen hours before BRET analysis cells were subjected or not to a Brefeldin A (BFA) treatment (see methods). (A) BFA-treated and untreated cells were stained for the Golgi marker Giantin; DAPI was added to label nuclei. Scale bar: 10μM. (B) FACS analysis of CCR5 cell surface expression in cells treated or not with BFA. The grey filled histogram is the isotypic control, the dotted open histogram corresponding to the specific signal (C) BRET analysis of CD4-CCR5 (wild-type and truncated) interaction in CHO-K1 cells after BFA treatment. Procedure and BRET controls were as in Figure 2E.

(A) CHO-K1 cells were transfected with the plasmid for CCR5-Luc with or without the plasmid for CD4-YFP at concentrations, which correspond to the asterisk in Figure 3B. Cells were serum-starved overnight and then stimulated with MIP1β (5×10⁻⁸M) for the indicated durations of time. The luciferase signal was measured to monitor the total amount of CCR5. Immunoblotting was performed with the indicated antibodies. Equal amounts of lysates were loaded in each lane and a representative immunoblot of 3 independent experiments is shown. Total ERK represents the loading control. (B) Quantification of phosphorylated ERK 1-2 (normalized with total ERK) from 3 independents experiments. * indicate significant difference from untreated cells; ‡ indicate significant difference between cells expressing or not CD4; * and ‡: p<0.05, **: p<0.01, *** and ‡‡‡: p<0.001.

(A) CHO-K1 cells transfected with HA-tagged CCR5-YFP or CCR5ΔCter-YFP were analyzed for surface expression by FACS using the 1/85a Alexa-fluor^R-647-conjugated anti-human CCR5, or (B) processed for immunofluorescence using the 3F10 anti-HA antibody. (C) Subcellular colocalization of CD4 with wild-type HA-CCR5-YFP and HA-CCR5ΔCter-YFP. CHO-K1 cells were co-transfected with CD4 and HA-CCR5-YFP or HA-CCR5ΔCter-YFP, permeabilized and stained for CD4 and calnexin using a monoclonal mouse anti-CD4 (OKT4) and goat anti-calnexin polyclonal antibody, respectively. After incubation with the appropriate Cy5 or Cy3-labeled secondary antibody, cells were examined with a confocal microscope. Note the substantial amount of intracellular CD4, likely due to the absence in CHO-K1 cells of the tyrosine kinase lck, which is known to stabilize CD4 at the cell surface of leukocytes. Scale bar: 10μM. (D) Co-immunoprecipitation experiments of CD4 with both wild-type and mutant CCR5ΔCter. CHO-K1 cells were co-transfected with plasmids coding for CD4 and HA-CCR5-YFP or HA-CCR5ΔCter or free YFP. After receptor precipitation with a monoclonal anti-GFP antibody, the presence of co-precipitated CD4 was revealed by immunoblot. (E) BRET analysis of CD4 interaction with wild-type and mutant CCR5. CHO-K1 cells were co-transfected with plasmids coding for CD4-Rluc (the BRET donor) and increasing concentrations of CCR5-YFP, CCR5ΔCter-YFP (the BRET acceptors) or GABABR1-YFP or free YFP (negative controls). Energy transfer was measured after addition of the membrane permeable luciferase substrate coelenterazine h. The BRET signal was determined by calculating the ratio of light emitted at 530nm over the light emitted at 485 nm as described in Material and Methods. BRET specificity was further controlled by showing that the BRET signal remained stable upon increasing the amounts of BRET pairs at a fixed ratio of donor: acceptor (data not shown). Maximal BRET signals were significantly different in cells expressing HA-CCR5ΔCter-YFP and HA-CCR5-YFP, likely because the variable length of the two C-terminal tails, to which the BRET acceptor was fused, affected the energy transfer. In contrast, the value of the YFP: Luc ratio, for which half-maximal BRET obtained, was comparable, indicating that these two forms of CCR5 display the same propensity to associate with CD4. The asterisk indicates the experimental conditions used for quantification of CD4 and CCR5 (see Sup. Figure 3A).

(A) CHO-K1 cells were co-transfected with fixed concentrations of a plasmid encoding CCR5-Luc and increasing concentrations of the CD4-YFP plasmid or controls. CCR5 surface expression was determined by FACS analysis using a mouse anti-human CCR5 (2D7) as primary antibody followed by a Cy5-conjugated anti-mouse antibody. Representative experiments are shown. Above each panel, the YFP-associated signal (between parenthesis) of the indicated YFP-fused protein is shown, using the same units as in panel B. (B) Results of 14 independent experiments, each representing 8–10 individual transfections. The luciferase signal was used to monitor the total amount of CCR5. Experiments in which the luciferase signal was different from the average by more than 10% were discarded. Surface CCR5 values, represented as the percentage (± SEM of 4–6 grouped values) of control cells expressing CCR5 alone, were plotted as a function of the YFP signal, reflecting the concentration of CD4-YFP, of control leptin receptor fused to YFP (OBR-YFP) or free YFP. The asterisk indicates the experimental conditions used for quantification of CD4 and CCR5 (see Sup Figure 3A). (C) Co-immunoprecipitation analysis of CD4 - CXCR4 interaction. CHO-K1 cells were co-transfected with plasmids coding for CD4 and CXCR4-YFP and processed for immunoprecipitation as described in Figure 2D. (D) Absence of CXCR4 cells surface modulation by increasing concentrations of CD4. The experiment was conducted as described in panel A, using a mouse anti-CXCR4 antibody (12G5) followed by a Cy5-conjugated anti-mouse antibody. Each point corresponds to an individual transfection.

(A) Western blot analysis of CD4 from THP-1 cells nucleofected with siRNAs directed against CD4 (S1, S2 and S3) or scrambled control. CD4 was detected using the 1F6 anti-human CD4 antibody. (B) Quantification of CD4 after treatment with indicated siRNAs (n=3, in triplicates). Asterisks indicate statistical significance (p<0.001) with cells treated with control siRNA. (C) FACS analysis of cell surface CCR5 using the 1/85a Alexa-fluor 647-conjugated anti-human CCR5. Grey open histograms of S1, S2 and S3-treated cells are compared to the control (scramble siRNA) histogram in black. Grey filled histogram corresponds to isotypic control. (D) The effect of CD4 inhibition on surface CCR5 was measured as follows: the proportion of surface CCR5 was calculated by FACS (by comparing the signal in unpermeabilzed and permeabilized cells) in 3 independent experiments, in triplicates (**p<0.01; ***p<0.001); the values were compared to the proportion of surface CCR5 in THP-1 nucleofected with control scramble siRNA. (E) Left panel: Representative western blot analysis of CD4 from donor-purified, activated (see methods) primary T cells, nucleofected with 2 siRNAs targeting CD4 sequences (S1 and S3); note that, as for THP-1 cells, the S3 siRNA did not reduce CD4 concentration in T cells. Right panel: effect of CD4 inhibition on surface CCR5 in T cells from 3 donors. The proportion of surface CCR5 was calculated by FACS, by comparing the signal in unpermeabilzed and permeabilized cells, and values obtained in cells nuleofected with the S1 siRNA were compared to the proportion of surface CCR5 in cells nucleofected with the ineffective S3 siRNA.